Abstract PTOA is caused by physical trauma such as sports related joint injuries. It is a common cause of joint degeneration and disability, affecting 5.6 millions of Americans every year. Currently, there is no disease- modifying drug to prevent PTOA progression. As a Nobel-prize winning discovery, small interfering RNA (siRNA) provides great therapeutic potential to specifically inhibit disease gene expression. However, it is extremely challenging to deliver negatively-charged siRNA to penetrate avascular, dense, negatively-charged cartilage matrix. Moreover, therapeutics in the joint capsule is usually cleared rapidly, limiting their residence times to as short as 1-5 hours. To overcome these obstacles, we developed a non-covalent Janus-base nano- delivery vehicle named Nanopiece (NP) with customized dimensions and surface properties, which enable the encapsulated siRNA to penetrate into articular cartilage tissue and enter chondrocytes. Through binding with extracellular matrix (ECM), NP is retained in cartilage for a long half-life, converting a barrier to carrier. The goal of this proposal is to determine the factors regulating the dimensions and surface properties of NPs, thereby 1) identifying the optimal dimensions of NPs that penetrate into cartilage effectively; 2) formulating surface properties of NPs that bind cartilage matrix for tissue retention, and 3) evaluating the therapeutic ability of the NPs to inhibit PTOA progression in the DMM model. The underlying rationale is that the completion of this proposal will 1) advance our understanding on the self-assembly of non-covalent nano-delivery vehicles and their interactions with tissue ECM molecules; 2) realize a platform siRNA delivery technology that penetrating cartilage and other matrix-rich tissues with customized dimensions and surface properties; and 3) lay the foundation for the development of the first disease-modifying RNA therapeutic against PTOA. The proposed research is innovative because: 1) NP is a new generation drug delivery vehicle with unique advantages such as the versatility in dimensions, affinity to ECM molecules and excellent biodegradability and non-toxicity. 2) We delineate the interactions between delivery vehicles and cartilage ECM in terms of the vehicles? dimension and surface property, and then determine the key factors for successful intra-cartilage delivery. 3) The technology breakthrough enlightens a therapeutic approach to deliver siRNA to treat PTOA. After accomplishing the specific aims, we expect to 1) advance fundamental understandings of the non- covalent nanomaterial self-assembly and its interactions with tissue matrix, 2) achieve highly effective and long-lasting siRNA delivery into cartilage, and 3) inhibit PTOA progression in the DMM model via the siRNA/NP therapy. These outcomes will have important positive impact on developing specific drug delivery vehicles for cartilage or other matrix-rich tissues, and improving treatment of joint diseases such as PTOA.